Monocyte-derived nucleic acids and related compositions and methods

ABSTRACT

Nucleic acids encoding various monocyte-derived proteins and related compositions, including purified proteins and specific antibodies are described. Methods of using such composition are also provided.

FIELD OF THE INVENTION

The present invention is directed to compositions related to genes foundin monocytes, cells which function in the immune system. These genesfunction in controlling development, differentiation, and/or physiologyof the mammalian immune system. In particular, the invention providesnucleic acids, proteins, antibodies, and methods of using them.

BACKGROUND OF THE INVENTION

Monocytes are phagocytic cells that belong to the mononuclear phagocytesystem and reside in the circulation. These cells originate in the bonemarrow and remain only a short time in the marrow compartment once theydifferentiate. They then enter the circulation and can remain there fora relatively long period of time, e.g., a few days. Monocytes can enterthe tissues and body cavities by a process known as diapedesis, wherethey differentiate into macrophages and possibly into dendritic cells.In an inflammatory response, the number of monocytes in the circulationmay double, and many of the increased number of monocytes diapedese tothe site of inflammation. For a review of monocytes and their functions,see, e.g., Gallin, et al. (eds), 1988, Inflammation: Basic Principlesand Clinical Correlates, Raven Press, NY; van Furth (ed), 1985,Mononuclear Phagocytes: Characteristics, Physiology and Function,Martinus Nijhoff, Dordrecht, Netherlands.

Antigen presentation refers to the cellular events in which aproteinaceous antigen is taken up, processed by antigen presenting cells(APC), and then recognized to initiate an immune response. The mostactive antigen presenting cells have been characterized as themacrophages, which are direct developmental products from monocytes;dendritic cells; and certain B cells.

Macrophages are found in most tissues and are highly active ininternalization of a wide variety of protein antigens andmicroorganisms. They have a highly developed endocytic activity, andsecrete many products important in the initiation of an immune response.For this reason, it is believed that many genes expressed by monocytesor induced by monocyte activation are important in antigen uptake,processing, presentation, or regulation of the resulting immuneresponse.

Despite the importance of monocytes to immune system function, thesecells remain poorly characterized, both in terms of the proteins theyexpress and in terms of many of their functions, in particular, theprocesses and mechanisms related to the initiation of an immuneresponse, including antigen processing and presentation. There is thus aneed in the art for agents useful in the diagnosis and treatment ofmedical conditions caused by, e.g., the inappropriate regulation,development, or physiology of antigen presenting cells.

SUMMARY OF THE INVENTION

The present invention fulfills this need by providing compositions andmethods for determining the presence, amount, distribution and normalcyof certain gene products and for facilitating the discovery of agentsfor treating certain disease states.

The invention is based upon the discovery of novel genes and geneproducts isolated from activated monocytes.

The invention provides isolated nucleic acid sequences comprising atleast about 12, preferably at least about 18, most preferably at leastabout 20-35, and most preferably 35-55 or more consecutive nucleotidesshown in SEQ ID NO: 1, 3, 5, 7, or 9, or which encode an amino acidsequence shown in SEQ ID NO: 2, 4, 6, 8 or 10, including completeprotein coding sequences, and complements thereof. The inventionencompasses sequence-conservative variants and function-conservativevariants of these sequences. The nucleic acids may be DNA, RNA, DNA/RNAduplexes, protein-nucleic acid (PNA), or derivatives thereof. Theinvention also encompasses recombinant DNA vectors (including DNAexpression vectors) comprising these sequences; cells comprising suchvectors, including bacterial, fungal, plant, insect, and mammaliancells; and methods for producing expression products comprising RNA andpolypeptides encoded by the sequences.

Polypeptide sequences of the invention comprise at least eight,preferably at least about 10, and more preferably at least about 12 ormore consecutive amino acid residues derived from SEQ ID NO: 2, 4, 6, 8or 10. Function-conservative variants and homologs are included in thescope of the invention.

The invention further provides binding compositions, in particularantibodies, most particularly monoclonal antibodies, which specificallybind to polypeptides having an amino acid sequence shown in SEQ ID NO:2, 4, 6, 8 or 10 or function conserved variants or homologs thereof.Methods are also provided for producing antibodies having the desiredbinding specificity in a host animal.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents, and literature references cited inthis specification are hereby incorporated herein by reference in theirentirety.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA, are used. Suchtechniques are well known and are explained fully in, for example,Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glovered.); Oligonucleotide Synthesis, 1984, (M. L. Gait ed.); Nucleic AcidHybridization, 1985, (Hames and Higgins); Transcription and Translation,1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (R. I. Freshneyed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, APractical Guide to Molecular Cloning; the series, Methods in Enzymology(Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987(J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory); andMethods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu,eds., respectively).

DEFINITIONS

1. A “monocyte-derived” nucleic acid or polypeptide refers to the sourcefrom which the sequence was originally isolated.

2. “Nucleic acid” or “polynucleotide” refers to purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides.This includes single- and double-stranded molecules, i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases.

3. A “coding sequence” or a “protein-coding sequence” is apolynucleotide sequence capable of being transcribed into mRNA and/orcapable of being translated into a polypeptide. The boundaries of thecoding sequence are typically determined by a translation start codon atthe 5′-terminus and a translation stop codon at the 3′-terminus.

4. A “complement” of a nucleic acid sequence refers to the “antisense”sequence that participates in Watson-Crick base-pairing with theoriginal sequence.

5. An “isolated” nucleic acid or polypeptide refers to component that isremoved from its original environment (for example, its naturalenvironment if it is naturally occurring). An isolated nucleic acid orpolypeptide preferably contains less than about 50%, more preferablyless than about 75%, and most preferably less than about 90%, of thecellular components with which it was originally associated.

6. A nucleic acid or polypeptide sequence that is “derived from” adesignated sequence refers to a sequence that corresponds to a region ofthe designated sequence. For nucleic acid sequences, this encompassessequences that are homologous or complementary to the sequence, as wellas “sequence-conservative variants” and “function-conservativevariants.” For polypeptide sequences, this encompasses“function-conservative variants.” Sequence-conservative variants arethose in which a change of one or more nucleotides in a given codonposition results in no alteration in the amino acid encoded at thatposition. Function-conservative variants are those in which a givenamino acid residue in a polypeptide has been changed withoutsubstantially altering the overall conformation and function of thenative polypeptide, including, but not limited to, replacement of anamino acid with one having similar physico-chemical properties (such as,for example, acidic, basic, hydrophobic, and the like).“Function-conservative” variants also include any polypeptides that havethe ability to elicit antibodies specific to a designated polypeptide.

7. A “probe” refers to a nucleic acid or oligonucleotide that forms ahybrid structure with a sequence in a target region due tocomplementarity of at least one sequence in the probe with a sequence inthe target.

8. Nucleic acids are “hybridizable” to each other when at least onestrand of nucleic acid can anneal to another nucleic acid strand underdefined stringency conditions. Stringency of hybridization isdetermined, e.g., by a) the temperature at which hybridization and/orwashing is performed, and b) the ionic strength and polarity (e.g.,formamide) of the hybridization and washing solutions, as well as otherparameters. Hybridization requires that the two nucleic acids containsubstantially complementary sequences; depending on the stringency ofhybridization, however, mismatches may be tolerated. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementarity, variables well known inthe art.

9. An “immunogenic component” is a moiety that is capable of eliciting ahumoral and/or cellular immune response in a host animal.

10. An “antigenic component” is a moiety that binds to its specificantibody with sufficiently high affinity to form a detectableantigen-antibody complex.

11. A “sample” refers to a biological sample, such as, for example,tissue or fluid isolated from an individual or from an in vitro cellculture constituents, as well as samples obtained from laboratoryprocedures.

The invention provides nucleic acid sequences encoding mammalianproteins expressed on monocytes. While specific human monocyte-derivedgenes and gene products are described herein, the invention encompassesstructurally (e.g., sequence) related embodiments from other sources ormammalian species, including polymorphic or individual variants. Thesewill include, e.g., proteins which exhibit relatively few changes insequence, e.g., less than about 5%, and number, e.g., less than 20residue substitutions, typically less than 15, preferably less than 10,and more preferably less than 5 substitutions. These will also includeversions which are truncated from full length and fusion proteinscontaining substantial segments of these sequences.

A gene/gene product, isolated from human monocyte cell library anddesignated FDF03, has been previously described in publishedInternational application WO 98/24906, the disclosure of which isincorporated herein in its entirety by reference. The FDF03 gene encodesa type I transmembrane protein comprising an extracellular portioncharacterized by Ig-like domains, indicating that this gene encodes areceptor member of the Ig superfamily.

SEQ NO: 1 shows the nucleic acid sequence encoding human FDF03 protein.The amino acid sequence of the FDF03 protein is shown in SEQ ID NO: 2.The putative coding region runs from about nucleotide 154 to nucleotide1062. An N-terminal hydrophobic sequence corresponding to a putaivesignal sequence runs from about amino acid residue-19 (Met) to aminoacid residue-1 (Leu). An internal hydrophobic sequence corresponding toa putative transmembrane segment runs from about residue 177 (Ala) toresidue 199 (Leu). The extracellular region is about 170 amino acids,with a potential Ig-like domain structure. The intracellular region isabout 80 residues. Sequence analysis indicates similarity to GenBankclones H26010 and R50327 from humans.

Four human FDF03 homologs have now been discovered.

ED F03-ΔTM

The second human clone, designated FDF03-delta™ (FDF03-ΔTM), appears tobe a soluble form of human FDF03 generated by alternative splicing. Thenucleic acid sequence encoding FDF03-ΔTM is shown in SEQ NO: 3. Theamino acid sequence of the FDF03-ΔTM protein is shown in SEQ ID NO: 4.

cDNA of the FDF03-ΔTM molecule was amplified along with that of FDF03during the analysis of human FDF03 expression by RT-PCR. Using primersdesigned in the 5′-UTR and 3′-UTR of FDF03 gene (FDF03-U25:5′-ACAGCCCTCTTC-GGAGCCTCA (SEQ ID NO: 11) and FDF03-L1166:5′-AAGCTGGCCCTGAACT CCTGG (SEQ ID NO: 12)), an approximately 200 basepair shorter band was amplified by RT-PCR from PMA/ionomycin activatedPBL cDNA, then gel purified, cloned and sequenced. Different clonescontained an identical insert of 943 base pairs with an open readingframe encoding a type I protein of 230 amino acids. The deduced aminoacid sequence of FDF03-ΔTM matched perfectly with that of FDF03, butcontained a gap of 73 amino acids that deleted the extracellularthreonine-rich region and the transmembrane domain of FDF03. Thisresulted in a protein with a potential hydrophobic signal peptidefollowed by the extracellular Ig like-domain linked to theintracytoplasmic domain of FDF03. cDNA alignments with FDF03 sequenceidentified a deletion of 219 nucleotides in the FDF03-ΔTM sequence(FDF03 nucleotide 608 to 827) that did not introduce premature stopcodons, suggesting that this molecule is the product of an alternativesplicing. This molecule is believed to be a secreted soluble form ofFDF03 and believed to bind to the same ligand(s) as FDF03.

The protein alignment of the FDF03 (SEQ ID NO: 2) and FDF03-ΔTM (SEQ IDNO: 4) is shown below.

1 MGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE FDF03 1MGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE FDF03-ΔTM61 LATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKSGFLRISNLQKQDQ FDF03 61LATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKSGFLRISNLQKQDQ FDF03-ΔTM121 SVYFCRVELDTRSSGRQQWQSIEGTKLSITQAVTTTTQRPSSMTTTWRLSSTTTTTGLRV FDF03121 SVYFCRVELDTRSSGRQQWQSIEGTKLSITQ-----------------------------FDF03-ΔTM 181TQGKRRSDSWHISLETAVGVAVAVTVLGIMILGLICLLRWRRRKGQQRTKATTPAREPFQ FDF03 152--------------------------------------------GQQRTKATTPAREPFQ FDF03-ΔTM241 NTEEPYENIRNEGQNTDPKLNPKDDGIVYASLALSSSTSPRAPPSHRPLKSPQNETLYSV FDF03168 NTEEPYENIRNEGQNTDPKLNPKDDGIVYASLALSSSTSPRAPPSHRPLKSPQNETLYSVFDF03-ΔTM 303 LKA FDF03 230 LKA FDF03-ΔTM (-:deletion)

FDF03-S1

The third clone, designated FDF03-Short 1 (FDF03-S1), is an Ig-likemolecule homologous to FDF03 but with a short intracytoplasmic domainand a charged residue in transmembrane domain. Comparative DNA andprotein analysis suggests the presence of different genes for FDF03 andFDF03S1, rather than alternatively spliced products. The nucleic acidsequence encoding FDF03-S1 is shown in SEQ NO: 5. The amino acidsequence of the FDF03-S1 protein is shown in SEQ ID NO: 6.

FDF03-S1 is a type I transmembrane protein belonging to the Igsuperfamily. FDF03-S1 contains a hydrophobic leader sequence followed byan extracellular region (˜170 residues) with a V-type Ig domainstructure homologous to that of FDF03 (88% homology at the amino acidlevel). Unlike FDF03, FDF03-S1 possesses a transmembrane domain with acharged amino acid (K), and a small intracellular tail (15 residues)without ITIM or internalization motif. FDF03-S1 is believed to representan activation isoform of FDF03 and may associate with ITIM-bearingmolecules such as DAP12. The amino acid sequence is shown below, whereinthe signal peptide and transmembrane domain are underlined. The chargedamino acid, lysine (K), residue (arrow) in the transmembrane domain maypermit association with another chain, for example DAP12.

(SEQ ID NO: 6) MGRPLLLPLLLLLQPPAFLQPGGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWELAIVPNVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQESGFLRISNLRKEDQSVYFCRVELDTRRSGRQQLQSIKGTKLTITQAVTTTTTWRPSSTTTIAGLRVTESKGHSESWHLSLDTAIRVALAVAVLKTVILGLLCLLLLWWRRRKGSRAPSSDF ↑

The protein alignment of FDF03 (SEQ ID NO: 2) and FDF03-S1 (SEQ ID NO:6) is shown below.

1 MGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE FDF03 1MGRPLLLPLLLLLQPPAFLQPGGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE FDF03-S1          +  +       + 61LATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKSGFLRISNLQKQDQ FDF03 61LAIVPNVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQESGFLRISNLRKEDQ FDF03-S1  ++ +                                       +         + + 121SVYFCRVELDTRSSGRQQWQSIEGTKLSITQAVTTTTQRPSSMTTTWRLSSTTTTTGLRV FDF03 121SVYFCRVELDTRRSGRQQLQSIKGTKLTITQAVTTTT........TWRPSSTTTIAGLRV FDF03-S1            +     +   +    +         ++++++++   +     ++ 181TQGKRRSDSWHISLETAVGVAVAVTVLGIMILGLICLL..RWRRRKGQQRTKATTPAREP FDF03 173TESKGHSESWHLSLDTAIRVALAVAVLKTVILGLLCLLLLWWRRRKGSRAPSSDF FDF03-S1 ++ ++ +   +  +  ++  +  +  +++    +   +++      ++++++++ 239FQNTEEPYENIRNEGQNTDPKLNPKDDGIVYASLALSSSTSPRAPPSHRPLKSPQNETLY FDF03 299SVLKA FDF03 +:residue different or gap

Distribution studies (RT-PCR) shows strong expression in B cells (poolresting+activated), T cells and PBL. Lower expression was observed inmonocytes, dendritic cells and granulocytes.

FDF03-M14

The fourth clone, desginated FDF03-M14, is a potential soluble form ofhuman FDF03 generated by alternative splicing. The nucleic acid sequenceencoding FDF03-M14 is shown in SEQ ID NO: 7. The amino acid sequence ofthe FDF03-M-14 protein is shown in SEQ ID NO: 8. cDNA of this moleculewas amplified along with that of FDF03 during the analysis of humanFDF03 expression by RT-PCR. Using primers designed in the 5′-UTR and3′-UTR of FDF03 gene (FDF03-U25: 5′-ACAGCCC-TCTTCGGAGCCTCA (SEQ ID NO:11) and FDF03-L1166: 5′-AGCTGGCCCTGA-ACTCCTGG (SEQ ID NO: 12)), anapproximately 200 base pair shorter band was amplified by RT-PCR fromactivated PBL cDNA, then gel purified, cloned and sequenced. One clone(M14) contained an insert of 908 base pairs with an ORF encoding a typeI protein of 175 amino acids. cDNA alignments with FDF03 sequenceidentified a deletion of 253 nucleotides in FDF03-M14 sequence (FDF03nucleotide 608 to 861) that deleted the sequences encoding theextracellular threonine-rich region, the transmembrane domain and thestart of the intracytoplasmic domain of FDF03, and that introduced aprematured stop codon at position 655 of FDF03-M14. The deduced aminoacid sequence of FDF03-M 14 resulted in a protein with a potentialhydrophobic signal peptide followed by an extracellular Ig like-domainthat matched perfectly with that of FDF03, but that was linked to aCOOH-terminal 24 amino acid sequence different from FDF03. This moleculemay be the product of an alternative splicing of FDF03 mRNA.

Like FDF03-ΔTM, this molecule may represent a secreted soluble form ofFDF03 and may bind to the same ligand(s) as FDF03. The amino acidsequence is shown below, wherein the signal sequence is underlined.

(SEQ ID NO: 8) MGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTUKHLSASMGGSVEIPFSFYYPWELATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKSGFLRISNLQKQDQSVYFCRVELDTRSSGRQQWQSIEGTKLSITQGNPSKTQRSHMRISGMRDKIQIPS

The protein alignment of FDF03 (SEQ ID NO: 2) and FDF03-M14 (SEQ ID NO:8) is shown below.

1 MGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE FDF03 1MGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE FDF03-14 61LATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKSGFLRISNLQKQDQ FDF03 61LATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKSGFLRISNLQKQDQ FDF03-M14121 SVYFCRVELDTRSSGRQQWQSIEGTKLSITQAVTTTTQRPSSMTTTWRLSSTTTTTGLRV FDF03121 SVYFCRVELDTRSSGRQQWQSIEGTKLSITQGNPSKTQRSHMRISGMRDKIQIPS FDF03-M14                               *****   ******** ******* 181TQGKRRSDSWHISLETAVGVAVAVTVLGIMILGLICLLRWRRRKGQQRTKATTPAREPFQ FDF03 241NTEEPYENIRNEGQNTDPKLNPKDDGIVYASLALSSSTSPRAPPSHRPLKSPQNETLYSVLKA FDF03*:residue different

FDF03-S2

The fifth clone, designated FDF03-S2 is an Ig-like molecule homologousto FDF03 but with a short intracytoplasmic domain and a charged residuein transmembrane domain. This molecule is highly homologous to FDF03-S1and is a potential DAP12-associated protein. The nucleic acid sequenceencoding FDF03-S2 is shown is SEQ ID NO: 9. The amino acid sequence ofthe FDF03-S2 protein is shown in SEQ ID NO: 10.

cDNA of this molecule was amplified using primers specific for FDF03-S2.Specificity is obtained with forward primer designed in 5′UTR ofFDF03-S2. FDF03-S2-forward: 5′-CAAGG-GATAAAAAGGCAC (SEQ ID NO: 13) (doesnot amplify FDF03, FDF03Δ™ or FDF03-S1). FDF03-S2-reverse:5′-AACTCTCCTCCAGTCGGT (SEQ ID NO: 14) (can amplify FDF03-S1, but notFDF03 or FDF03delta™).

FDF03-S2 is a type I transmembrane protein belonging to the Igsuperfamily. FDF03-S2 contains a hydrophobic leader sequence followed byan extracellular region (˜170 residues) with one V-type Ig domainstructure homologous to that of FDF03 (˜85% homology at the amino acidlevel). Unlike FDF03, FDF03-S2 possesses a transmembrane domain with acharged amino acid (K), and a small intracellular tail (15 residues)without ITIM or internalization motif. FDF03-S2 is highly homologous toFDF03-S1 (3 amino acid difference in the extracellular domain and oneamino acid missing in the transmembrane domain). Like FDF03-S1, FDF03-52may represent an activation isoform of FDF03 and may associate withITAM-bearing molecules such as DAP12.

There are two putative start codons in frame (position 117 and 309). Thefirst one is not contained within a typical Kozak sequence. The sequenceshown below is deduced from the second start codon (nucleotide 309), asstarting at the first start codon in frame (position 117) does notencode for a hydrophobic sequence followed by another Ig-like domain. Inthe sequence shown below, the signal peptide and transmembrane domainare underlined. The charged amino acid, lysine (K) residue (arrow) intransmembrane domain may permit association with another chain, forexample DAP12.

MGRPLLLPLLLLLQPPAFLQPGGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWELATAPDVRISWRRGHFHGQSFYSTRPPSIHEDYVNRLFLNWTEGQESGFLRISNLRKEDQSVYFCRVELDTRRSGRQQLQSIKGTKLTITQAVTTTTTWRPSSTTTIAGLRVTESKGHSESWHLSLDTAIRVALAVAVLKTVILGLLCLLLWWRRRKGSRAPSSDF ↑

The protein alignments of FDF03, FDF03-S1 and FDF03-S2 is shown below.

1 MGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE FDF03 1MGRPLLLPLLLLLQPPAFLQPGGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE FDF03-S1 1MGRPLLLPLLLLLQPPAFLQPGGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE FDF03-S2          +  +       + 61LATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKSGFLRISNLQKQDQ FDF03 61LAIVPNVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQESGFLRISNLRKEDQ FDF03-S1 61LATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQESGFLRISNLRKEDQ FDF03-S2** *                                       +         + + 121SVYFCRVELDTRSSGRQQWQSIEGTKLSITQAVTTTTQRPSSMTTTWRLSSTTTTTGLRV FDF03 121SVYFCRVELDTRRSGRQQLQSIKGTKLTITQAVTTTT........TWRPSSTTTIAGLRV FDF03-S1121 SVYFCRVELDTRRSGRQQLQSIKGTKLTITQAVTTTT........TWRPSSTTTIAGLRVFDF03-S2             +     +   +    +         ++++++++   +     ++ 181TQGKRRSDSWHISLETAVGVAVAVTVLGIMILGLICLL..RWRRRKGQQRTKATTPAREP FDF03 173TESKGHSESWHLSLDTAIRVALAVAVLKTVILGLLCLLLLWWRRRKGSRAPSSDF FDF03-S1 173TESKGHSESWHLSLDTAIRVALAVAVLKTVILGLLCLLL.WWRRRKGSRAPSSDF FDF03-S2 ++ ++ +   +  +  ++  +  +  +++    +   +*+      ++++++++ 239FQNTEEPYENIRNEGQNTDPKLNPKDDGIVYASLALSSSTSPRAPPSHRPLKSPQNETLY FDF03 299SVLKA FDF03 +:residue different or gap between FDF03-S2/FDF03-S1 andFDF03 *:residue different or gap between FDF03-S2/FDF03 and FDF03-S1

Distribution studies (RT-PCR) shows expression in activated dendriticcells (CD34-derived), PBMC, monocytes and tonsil B cells.

Alignment with human IgV domains and TCR V domain is given below. Thisalignment shows the conserved VDJ structure of FDF03.

Ig V region QVQ.LQESGPG.LVKPSETLSLTCTVSGGSVSSGSYYWSW.IRQAPGKGLEWIG TCRhuman QVQ.LQESGPG.LVKPSETLSLTCTVSGYSISSG.YYWGW.IRQPPGKGLEWIG FDF03QPSGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWELATAPDVRISWRR FDF03-S1 QPGGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWELAIVPNVRISWRR FDF03-S2 QPGGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWELATAPDVRISWRR+      +++  +        +     +        +  +     +     + Ig V regionYIYYSGSTNY.......NRSHKSRVNIS.VDTAKNQFSLKLSSVSTADTAVYYCARIT TCR humanSIYHSGSTYY.......NPSLKSRVTIS.VDTSKNQFSLKLSSVTAADTAVYYCARVR FDF03GHFH.GQSFYSTRPPSIHKDYVNALFLNWTEGQKSGF.LRISNLQKQDQSVYFC.RVE FDF03-S1 GHFH.GQSFYSTRPPSIHKDYVNRLFLNWTEGQESGF.LRISNLRKEDQSVYFC.RVE FDF03-S2 GHFH.GQSFYSTRPPSIHKDYVNRLFLNWTEGQESGF.LRISNLRKEDQSVYFC.RVE     +   +             +            + +  +     +  ++ + + Ig V regionTTVPSSWYYYYMDVWDKGTTVTVSS TCR human RRYSSSAS...KIIFGSGTRLSIR. FDF03 LDTRSSGRQQWQS..IEGTKLSITQ FDF03-S1  LDTRRSGRQQLQS..IKGTKLTITQ FDF03-S2 LDTRRSGRQQLQS..IKGTKLTITQ      +           ++

Studies of human genomic DNA clones show that chromosome 7 contains bothFDF03-S1 and FDF03 specific sequences, confirming that the two moleculesare encoded by two different genes. These studies also suggest thatFDF03-S1 and -S2 genes are two different alleles of the same gene. Inaddition, PCR from intronic sequence surrounding the areas of differencebetween S1 and S2 on genomic DNA from different donors shows theexistence of homozygotes and S1/S2 heterozygotes at this locus. It isthus likely that these two cDNAs are from different alleles.

The genomic organization of the FDF03 gene confirms that FDF03-ΔTM isproduced by alternative splicing (deletion of exon 3 coding for thehinge region and TM domain). This is also the case for FDF03-M14(deletion of exons 3 and 4).

The two forms of FDF03-S1/2 may be advantageously used as populationmarkers. The two forms of this protein will either not bind the sameligand (e.g., as in the case of the NK receptor family) or will bind atdifferent affinities, thus potentially giving individuals a differentresponse to receptor/ligand interaction.

The localization of the genes encoding FDF03 (including the ATM and M14forms) and FDF03-S1 on human chromosome 7q22 is interesting because thisregion is frequently deleted in myelodystrophic syndromes such as AcuteMyeloid Leukemia (AML). The implication of the possible deletion of amyeloid inhibitory receptor in a proliferative disease leads to apossible use in gene therapy.

Nucleic Acids, Vectors, and Host Cells

The invention provides nucleic acid sequences, in particular the nucleicacid sequences shown in SEQ ID NO: 1, 5, 7 or 9 or nucleic acidsequences which encode an amino acid sequences shown in SEQ ID NO: 2, 4,6, 8 or 10. The invention encompasses isolated nucleic acid fragmentscomprising all or part of the individual nucleic acid sequencesdisclosed herein. The nucleic acid sequences of the invention compriseat least about 12, preferably at least about 18, more preferably atleast about 20-35 and most preferably at least about 35-55 or moreconsecutive nucleotides, including complete protein-coding sequences, orcomplements thereof. The invention encompasses sequence-conservativevariants and function-conservative variants of these sequences.

Nucleic acids comprising any of the sequences disclosed herein orsubsequences thereof can be prepared by standard methods using thenucleic acid sequence information provided in SEQ ID NO: 1, 3, 5, 7 and9. For example, nucleic acids can be chemically synthesized using, e.g.,the phosphoramidite solid support method of Matteucci et al., 1981, J.Am. Chem. Soc. 103:3185, the method of Yoo et al., 1989, J. Biol. Chem.764:17078, or other well known methods. This can be done by sequentiallylinking a series of oligonucleotide cassettes comprising pairs ofsynthetic oligonucleotides. The nucleic acids may be isolated directlyfrom cells. Alternatively, the polymerase chain reaction (PCR) methodcan be used to produce the nucleic acids of the invention, using eitherchemically synthesized strands or genomic material as templates. Primersused for PCR can be synthesized using the sequence information providedherein and can further be designed to introduce appropriate newrestriction sites, if desirable, to facilitate incorporation into agiven vector for recombinant expression. Of course, due to thedegeneracy of the genetic code, many different nucleotide sequences canencode polypeptides having the amino acid sequences defined by SEQ IDNO: 2, 4, 6, 8 or 10 subsequences thereof. The codons can be selectedfor optimal expression in prokaryotic or eukaryotic systems. Suchdegenerate variants are also encompassed by this invention.

The encoded polypeptides may be expressed by using many known vectorssuch as pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), orpRSET or pREP (Invitrogen, San Diego, Calif.), and many appropriate hostcells such as Escherichia coli, Saccharomyces cerevisiae, and insect andmammalian cell lines using methods known to those skilled in the art.The particular choice of vector/host is not critical to the practice ofthe invention.

The nucleic acids of the present invention find use, e.g., as templatesfor the recombinant production of peptides or polypeptides, as probesand primers for the detection of the human genes described herein, forchromosome mapping, and as probes or to design PCR primers to identifyhomologous genes in other mammalian species. Homology may be determinedexperimentally. Alternatively, homology analysis may be performedcomputationally. In practicing the present invention, a gene that sharesat least about 70% DNA sequence homology at the nucleotide level withthe genome of another mammalian species is considered to be present inthat species. The determination that a gene is present in another mammalmay be achieved using any technique known in the art. Appropriatetechniques include without limitation hybridization to genomic DNA,colony hybridization to a genomic or cDNA library, polymerase chainreaction (PCR) using degenerate primers or gene-specific primers andgenomic DNA as a template, genetic complementation, antibodycross-reactivity, or biochemical complementation in vitro.

In applying these techniques, conditions are established thatdiscriminate different levels of homology between probe and template.For example, for hybridization of a probe to immobilized DNA (whether ina Southern blot, dot blot, or colony hybridization format), varying theSSC concentration in the buffer allows the detection of hybrids havingdifferent levels of homology (1×SSC is 0.15 M NaCl-0.015 M Na citrate).In a wash buffer containing 6M urea and 0.4% sodium dodecyl sulfate, thepresence of 2×SSC, 0.5×SSC, 0.1×SSC, and 0.05×SSC allows the formationof hybrids having threshold homologies of at least 55%+5%, 65%+5%,75%+5%, and >85%, respectively. Preferably, once a gene has beenidentified in another organism by hybridization or PCR, the DNA sequenceof the gene is determined directly.

It will be understood that some methods that detect homologous sequencesmay result in the identification or isolation of only a portion of theentire protein-coding sequence of a particular gene. The entireprotein-coding sequence can be isolated and identified, for example, byusing an isolated nucleic acid encoding the known portion of thesequence, or fragments thereof, to prime a sequencing reaction with cDNAas template; this is followed by sequencing the amplified product. Theisolated nucleic acid encoding the disclosed sequence, or fragmentsthereof, can also be hybridized to appropriate cDNA libraries toidentify clones containing additional complete segments of theprotein-coding sequence of which the shorter sequence forms a part.Then, the entire protein-coding sequence, or fragments thereof, ornucleic acids encoding all or part of the sequence, orsequence-conservative or function-conservative variants thereof, may beemployed in practicing the present invention.

In a similar manner, additional sequences derived from the 5′ and 3′flanking regions of sequence encoding the protein, including regulatorysequences, may be isolated, and the nucleotide sequence determined.

Polypeptides

Both the naturally occurring and recombinant forms of the polypeptidesdescribed herein, including both glycosylated and non-glycosylated formsare encompassed by the invention. The polypeptides of the presentinvention, including function-conservative variants, may be isolatedfrom human monocytes, or from heterologous organisms or cells (e.g.,bacteria, fungi, insect, plant, and mammalian cells) into which aprotein-coding sequence has been introduced and expressed. The proteinsdescribed herein, or portions thereof, also may be expressed as fusionswith other proteins. The polypeptides may be chemically synthesized bycommercially available automated procedures, including, withoutlimitation, exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thepolypeptides can also, advantageously, be made by in vitro translation.

Methods for polypeptide purification are well-known in the art,including, without limitation, preparative disc-gel electrophoresis,isoelectric focusing, sucrose density gradient centrifugation, HPLC,reversed-phase HPLC, gel filtration, ion exchange and partitionchromatography, and countercurrent distribution. For some purposes, itis preferable to produce the polypeptide in a recombinant system inwhich the protein contains an additional sequence tag that facilitatespurification, such as, but not limited to, a polyhistidine sequence. Thepolypeptide can then be purified from a crude lysate of the host cell bychromatography on an appropriate solid-phase matrix. Alternatively,antibodies produced against a protein or against peptides derivedtherefrom can be used as purification reagents. Other purificationmethods are possible.

The present invention also encompasses derivatives and homologues of thepolypeptides specifically disclosed herein. For some purposes, nucleicacid sequences encoding the peptides may be altered by substitutions,additions, or deletions that provide for functionally equivalentmolecules, i.e., function-conservative variants. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of similar properties, such as, for example,positively charged amino acids (arginine, lysine, and histidine);negatively charged amino acids (aspartate and glutamate); polar neutralamino acids; and non-polar amino acids.

The isolated polypeptides may be modified by, for example,phosphorylation, sulfation, acylation, or other protein modifications.They may also be modified with a label capable of providing a detectablesignal, either directly or indirectly, including, but not limited to,radioisotopes and fluorescent compounds.

The polypeptides of the invention find use, e.g., for binding studies,for construction and expression of modified molecules, forstructure/function studies and for the preparation of polyclonal andmonoclonal antibodies. Polypeptides useful as immunogenic components forpreparing antibodies or as targets for binding agent studies are atleast five or more residues in length. Preferably, the polypeptidescomprise at least about 12, more preferably at least about 20, and mostpreferably at least about 30 or more residues. Methods for obtainingthese polypeptides are well known and are explained in ImmunochemicalMethods in Cell and Molecular Biology, 1987 (Mayer and Waler, eds;Academic Press, London); Scopes, 1987, Protein Purification: Principlesand Practice, Second Edition (Springer-Verlag, N.Y.) and Handbook ofExperimental Immunology, 1986, Volumes I-IV (Weir and Blackwell, eds.).

Having isolated one member of a binding partner of a specificinteraction, methods exist for isolating the counter-partner. See, e.g.,Gearing et al., 1989, EMBO J. 8:3667-3676. Many methods of screening forbinding activity are known by those skilled in the art and may be usedto practice the invention. For example, an expression library can bescreened for specific binding to the protein, e.g., by cell sorting, orother screening to detect subpopulations which express such a bindingcomponent. See, e.g., Ho et al., 1993, Proc. Natl. Acad. Sci. USA90:11267-11271. Alternatively, a panning method may be used. See, e.g.,Seed and Aruffo, 1987, Proc. Natl. Acad. Sci. USA 84:3365-3369. Atwo-hybrid selection system may also be applied making appropriateconstructs with the available protein sequences. See, e.g., Fields andSong, 1989, Nature 340:245-246. Several methods of automated assays havebeen developed in recent years so as to permit screening of tens ofthousands of compounds in a short period of time.

Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence similarity with an amino acid sequence of a SEQ IDNO: 2, 4, 6, 8 or 10. Variants exhibiting substitutions, e.g., 20 orfewer, preferably 10 or fewer, and more preferably 5 or fewersubstitutions, are encompassed. Where the substitutions are conservativesubstitutions, the variants will share immunogenic or antigenicsimilarity or cross-reactivity with a corresponding natural sequenceprotein. Natural variants include individual, allelic, polymorphic,strain, or species variants.

Amino acid sequence similarity, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. This changes when considering conservative substitutions asmatches. Conservative substitutions typically include substitutionswithin the following groups: glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. Homologousamino acid sequences include natural allelic and interspecies variationsin each respective protein sequence. Typical homologous proteins orpeptides will have from 50-100% similarity (if gaps can be introduced),to 75-100% similarity (if conservative substitutions are included) withthe amino acid sequence of the relevant protein. Identity measures willbe at least about 50%, generally at least 60%, more generally at least65%, usually at least 70%, more usually at least 75%, preferably atleast 80%, and more preferably at least 80%, and in particularlypreferred embodiments, at least 85% or more. See also Needleham et al.,1970, J Mol. Biol. 48:443-453; Sankoff et al., 1983, Time Warps, StringEdits, and Macromolecules: The Theory and Practice of SequenceComparison Chapter One, Addison-Wesley, Reading, Mass.; and softwarepackages from IntelliGenetics, Mountain View, Calif.; and the Universityof Wisconsin Genetics Computer Group (GCG), Madison, Wis.

Nucleic acids encoding the corresponding proteins will typicallyhybridize to SEQ ID NO: 1, 3, 5, 7 or 9 under stringent conditions. Forexample, nucleic acids encoding the respective proteins will typicallyhybridize to the nucleic acid of SEQ ID NO: 1, 3, 5, 7 or 9 understringent hybridization conditions, while providing few false positivehybridization signals. Generally, stringent conditions are selected tobe about 10° C. lower than the thermal melting point (Tm) for thesequence being hybridized to at a defined ionic strength and pH. The Tmis the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration inwash is about 0.02 molar at pH 7 and the temperature is at least about50° C. Other factors may significantly affect the stringency ofhybridization, including, among others, base composition and size of thecomplementary strands, the presence of organic solvents such asformamide, and the extent of base mismatching. A preferred embodimentwill include nucleic acids that will bind to disclosed sequences in 50%formamide and 20-50 mM NaCl at 42° C.

An isolated nucleic acid can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of nucleotide stretches. These modifications result in novelDNA sequences which encode these antigens, their derivatives, orproteins having highly similar physiological, immunogenic, or antigenicactivity.

Modified sequences can be used to produce mutant antigens or to enhanceexpression. Enhanced expression may involve gene amplification,increased transcription, increased translation, and other mechanisms.Such mutant protein derivatives include predetermined or site-specificmutations of the respective protein or its fragments. “Mutant protein”encompasses a polypeptide otherwise falling within the homologydefinition of the proteins as set forth above, but having an amino acidsequence which differs from that of the protein as found in nature,whether by way of deletion, substitution, or insertion. In particular,“site specific mutant protein” generally includes proteins havingsignificant similarity with a protein having a sequence of SEQ ID NO: 2,4, 6, 8 or 10. Generally, the variant will share many physicochemicaland biological activities, e.g., antigenic or immunogenic, with thosesequences, and in preferred embodiments contain most or all of thedisclosed sequence.

Glycosylation alterations are included, e.g., made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing, or in further processing steps. Particularly preferred meansfor accomplishing this are by exposing the polypeptide to glycosylatingenzymes derived from cells that normally provide such processing, e.g.,mammalian glycosylation enzymes. Deglycosylation enzymes are alsocontemplated. Also embraced are versions of the same primary amino acidsequence which have other minor modifications, including phosphorylatedamino acid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine, or other moieties, including ribosyl groups orcross-linking reagents. Also, proteins comprising substitutions areencompassed, which should retain substantial immunogenicity, to produceantibodies that recognize a protein of SEQ ID NO: 2, 4, 6, 8 or 10.Typically, these proteins will contain less than 20 residuesubstitutions from the disclosed sequence, more typically less than 10substitutions, preferably less than 5, and more preferably less thanthree. Alternatively, proteins that begin and end at structural domainswill usually retain antigenicity and cross immunogenicity.

A major group of derivatives are covalent conjugates of the proteinsdescribed herein or fragments thereof with other proteins orpolypeptides. These derivatives can be synthesized in recombinantculture such as N- or C-terminal fusions or by the use of agents knownin the art for their usefulness in cross-linking proteins throughreactive side groups. Preferred protein derivatization sites withcross-linking agents are at free amino groups, carbohydrate moieties,and cysteine residues.

Fusion polypeptides between these proteins and other homologous orheterologous proteins are also provided. Heterologous polypeptides maybe fusions between different surface markers, resulting in, e.g., ahybrid protein. Likewise, heterologous fusions may be constructed whichwould exhibit a combination of properties or activities of thederivative proteins. Typical examples are fusions of a reporterpolypeptide, e.g., luciferase, with a segment or domain of a protein,e.g., a receptor-binding segment, so that the presence or location ofthe fused protein may be easily determined. See, e.g., U.S. Pat. No.4,859,609. Other gene fusion partners include bacterial β-galactosidase,trpE, Protein A, β-lactamase, alpha amylase, alcohol dehydrogenase, andyeast alpha mating factor. See, e.g., Godowski et al., 1988, Science241:812-816.

Such polypeptides may also have amino acid residues that have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those that havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets, e.g., affinity ligands.

This invention also contemplates the use of derivatives of theseproteins other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties. These derivatives generally fall into the threeclasses: (1) salts, (2) side chain and terminal residue covalentmodifications, and (3) adsorption complexes, for example with cellmembranes. Such covalent or aggregative derivatives are useful asimmunogens, as reagents in immunoassays, or in purification methods suchas for affinity purification of ligands or other binding ligands. Forexample, a protein antigen can be immobilized by covalent bonding to asolid support such as cyanogen bromide-activated Sepharose, by methodswhich are well known in the art, or adsorbed onto polyolefin surfaces,with or without glutaraldehyde cross-linking, for use in the assay orpurification of antibodies. The proteins can also be labeled with adetectable group, e.g., radioiodinated by the chloramine T procedure,covalently bound to rare earth chelates, or conjugated to anotherfluorescent moiety for use in diagnostic assays. Purification of theseproteins may be accomplished by immobilized antibodies.

Antibodies

The immunogenic components of this invention, as described above, areuseful as antigens for preparing antibodies by standard methods. Suchimmunogenic components can be produced by proteolytic cleavage of largerpolypeptides or by chemical synthesis or recombinant technology and arethus not limited by proteolytic cleavage sites. Preferably, smallerimmunogenic components will first be rendered more immunogenic bycross-linking or by coupling to an immunogenic carrier molecule (i.e., amacromolecule having the property of independently eliciting animmunological response in a host animal, to which the immunogeniccomponents of the invention can be covalently linked). Cross-linking orconjugation to a carrier molecule may be required because smallpolypeptide fragments sometimes act as haptens (molecules which arecapable of specifically binding to an antibody but incapable ofeliciting antibody production, i.e., they are not immunogenic).Conjugation of such fragments to an immunogenic carrier molecule rendersthem immunogenic through what is commonly known as the “carrier effect”.

Antibodies according to the present invention include polyclonal andmonoclonal antibodies. The antibodies may be elicited in an animal hostby immunization with immunogenic components of the invention or may beformed by in vitro immunization (sensitization) of immune cells. Theimmunogenic components used to elicit the production of antibodies maybe isolated from human cells (e.g., human monocytes) or chemicallysynthesized. The antibodies may also be produced in recombinant systemsprogrammed with appropriate antibody-encoding DNA. Alternatively, theantibodies may be constructed by biochemical reconstitution of purifiedheavy and light chains.

The antibodies of this invention can be purified by standard methods,including but not limited to preparative disc-gel electrophoresis,isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ionexchange and partition chromatography, and countercurrent distribution.Purification methods for antibodies are disclosed, e.g., in The Art ofAntibody Purification, 1989, Amicon Division, W.R. Grace & Co. Generalprotein purification methods are described in Protein Purification.Principles and Practice, R. K. Scopes, Ed., 1987, Springer-Verlag, NewYork, N.Y.

Suitable adjuvants for the vaccination of animals include but are notlimited to Adjuvant 65 (containing peanut oil, mannide monooleate andaluminum monostearate); Freund's complete or incomplete adjuvant;mineral gels such as aluminum hydroxide, aluminum phosphate and alum;surfactants such as hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecyl-ammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl) propane-diamine,methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran,dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides suchas muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.The immunogenic components could also be administered followingincorporation into liposomes or other microcarriers. Informationconcerning adjuvants and various aspects of immunoassays are disclosed,e.g., in the series by P. Tijssen, 1987, Practice and Theory of EnzymeImmunoassays, 3rd Edition, Elsevier, N.Y.

Serum produced from animals thus immunized can be used directly.Alternatively, the IgG fraction can be separated from the serum usingstandard methods such as plasmaphoresis or adsorption chromatographyusing IgG specific adsorbents such as immobilized Protein A.

Hybridomas of the invention used to make monoclonal antibodies againstthe immunogenic components of the invention are produced by well-knowntechniques. Usually, the process involves the fusion of an immortalizingcell line with a B-lymphocyte that produces the desired antibody.Alternatively, non-fusion techniques for generating immortalantibody-producing cell lines are possible, and come within the purviewof the present invention, e.g., virally-induced transformation, Casaliet al., 1986, Science 234:476. Immortalizing cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine, and human origin. Most frequently, rat or mouse myeloma celllines are employed as a matter of convenience and availability.

Techniques for obtaining the appropriate lymphocytes from mammalsinjected with the immunogenic components are well known. Generally,peripheral blood lymphocytes (PBLs) are used if cells of human originare desired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. A host animal is injected with repeateddosages of a preferably purified immunogenic component, and the animalis permitted to generate the desired antibody-producing cells beforethese are harvested for fusion with the immortalizing cell line.Techniques for fusion are also well known in the art, and in generalinvolve mixing the cells with a fusing agent, such as polyethyleneglycol.

Hybridomas are selected by standard procedures, such as HAT(hypoxanthine-aminopterin-thymidine) selection. From among thesehybridomas, those secreting the desired antibody are selected byassaying their culture medium by standard immunoassays, such as Westernblotting, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), or the like. Antibodies are recovered from themedium using standard protein purification techniques, Tijssen, 1985,Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam.

Many references are available for guidance in applying any of the abovetechniques: Kohler et al., 1980,Hybridoma Techniques, Cold Spring HarborLaboratory, New York; Tijssen, 1985, Practice and Theory of EnzymeImmunoassays, Elsevier, Amsterdam; Campbell, 1984, Monoclonal AntibodyTechnology, Elsevier, Amsterdam; Hurrell, 1982, Monoclonal HybridomaAntibodies: Techniques and Applications, CRC Press, Boca Raton, Fla.Monoclonal antibodies can also be produced using well known phagelibrary systems.

The use and generation of antibody fragments is also well known, e.g.,Fab fragments: Tijssen, 1985, Practice and Theory of EnzymeImmunoassays, Elsevier, Amsterdam; Fv fragments: Hochman et al., 1973,Biochemistry 12:1130; Sharon et al., 1976, Biochemistry 15:1591; Ehrlichet al., U.S. Pat. No. 4,355,023; and antibody half molecules:Auditore-Hargreaves, U.S. Pat. No. 4,470,925. These also may be usefulin immunoassays.

These antibodies, whether polyclonal or monoclonal, can be used, e.g.,in an immobilized form bound to a solid support by well known methods,to isolate and purify the immunogenic components by immunoaffinitychromatography. The antibodies are useful as probes to distinguishtissue and cell type distribution. The antibodies may be used to screenexpression libraries for particular expression products. Usually theantibodies used in such a procedure will be labeled with a moietyallowing easy detection of presence of antigen by antibody binding.Antibodies to proteins may be used for the analysis or, oridentification of specific cell population components which express therespective protein. By assaying the expression products of cellsexpressing the proteins described herein it is possible to diagnosedisease, e.g., immune-compromised conditions, monocyte depletedconditions, or overproduction of monocytes. Antibodies raised againstthe proteins will also be useful to raise anti-idiotypic antibodies.These will be useful in detecting or diagnosing various immunologicalconditions related to expression of the respective antigens. The presentinvention encompasses antibodies that specifically recognizemonocyte-derived immunogenic components. Such antibodies can be usedconventionally, e.g., as reagents for purification of monocyte cellcomponents, or in diagnostic applications.

Diagnostic Applications

The invention encompasses compositions, methods, and kits useful inclinical settings for the qualitative or quantitative diagnosis, i.e.,detection of specific components in a biological sample. Theseapplications utilize nucleic acids, peptides/polypeptides, or antibodiesspecific for the components described herein. Both antibody-based andnucleic acid-based diagnostic methods, including PCR-based diagnosticmethods are contemplated. Detection of the level of monocyte cellspresent in a sample is important for diagnosis of certain aberrantdisease conditions. For example, an increase in the number of monocytesin a tissue or the lymph system can be indicative of the presence of amonocyte hyperplasia, tissue or graft rejection, or inflammation. A lowmonocyte population can indicate an abnormal reaction to, e.g., abacterial or viral infection, which may require an appropriate treatmentto normalize the monocyte response.

Both the naturally occurring and the recombinant form of the proteins ofthis invention are particularly useful in kits and assay methods whichare capable of screening compounds for binding activity to the proteins.

In nucleic-acid-type diagnostic methods, the sample to be analyzed maybe contacted directed with the nucleic acid probes. Probes includeoligonucleotides at least 12 nucleotides, preferably at least 18, andmost preferably 20-35 or more nucleotides in length. Alternatively, thesample may be treated to extract the nucleic acids contained therein. Itwill be understood that the particular method used to extract DNA willdepend on the nature of the biological sample. The resulting nucleicacid from the sample may be subjected to gel electrophoresis or othersize separation techniques, or, the nucleic acid sample may beimmobilized on an appropriate solid matrix without size separation orused for PCR.

Kits suitable for antibody-based diagnostic applications typicallyinclude one or more of the following components:

(i) Antibodies: The antibodies may be pre-labeled; alternatively, theantibody may be unlabelled and the ingredients for labeling may beincluded in the kit in separate containers, or a secondary, labeledantibody is provided; and

(ii) Reaction components: The kit may also contain other suitablypackaged reagents and materials needed for the particular immunoassayprotocol, including solid-phase matrices, if applicable, and standards.

Kits suitable for nucleic acid-based diagnostic applications typicallyinclude the following components:

(i) Probe DNA: The probe DNA may be pre-labeled; alternatively, theprobe DNA may be unlabelled and the ingredients for labeling may beincluded in the kit in separate containers; and

(ii) Hybridization reagents: The kit may also contain other suitablypackaged reagents and materials needed for the particular hybridizationprotocol, including solid-phase matrices, if applicable, and standards.

PCR based diagnostic kits are also contemplated and are encompassed bythe invention.

The kits referred to above may include instructions for conducting thetest. Furthermore, in preferred embodiments, the diagnostic kits areadaptable to high-throughput and/or automated operation.

Therapeutic Applications

The invention also provides reagents that may exhibit significanttherapeutic value. The proteins (naturally occurring or recombinant),fragments thereof, and antibodies thereto, along with compoundsidentified as having binding affinity to the proteins, may be useful inthe treatment of conditions associated with abnormal physiology ordevelopment. For example, a disease or disorder associated with abnormalexpression or abnormal signaling by a monocyte, e.g., as an antigenpresenting cell, is a target for an agonist or antagonist of theprotein. The proteins likely play a role in regulation or development ofhematopoietic cells, e.g., lymphoid cells, which affect immunologicalresponses, e.g., antigen presentation and the resulting effectorfunctions.

Other abnormal developmental conditions are known in cell types shown topossess monocyte protein mRNA by northern blot analysis. See Berkow(ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway,N.J.; and Thorn, et al. Harrison's Principles of Internal Medicine,McGraw-Hill, NY. Developmental or functional abnormalities, e.g., of theimmune system, cause significant medical abnormalities and conditionsthat may be susceptible to prevention or treatment using compositionsprovided herein.

Recombinant monocyte-derived proteins or antibodies of the invention maybe purified and then administered to a patient. These reagents can becombined for therapeutic use with additional active or inertingredients, e.g., in conventional pharmaceutically acceptable carriersor diluents, e.g., immunogenic adjuvants, along with physiologicallyinnocuous stabilizers and excipients. In particular, these may be usefulin a vaccine context, where the antigen is combined with one of thesetherapeutic versions of agonists or antagonists. These combinations canbe sterile filtered and placed into dosage forms as by lyophilization indosage vials or storage in stabilized aqueous preparations. Thisinvention also contemplates use of antibodies or binding fragmentsthereof, including forms which are not complement binding.

Drug screening using antibodies or receptor or fragments thereof canidentify compounds having binding affinity to these monocyte-derivedproteins, including isolation of associated components. Subsequentbiological assays can then be utilized to determine if the compoundblocks or antagonizes the activity of the protein. Likewise, a compoundhaving intrinsic stimulating activity might activate the cell throughthe protein and is thus an agonist. This invention further contemplatesthe therapeutic use of antibodies to the proteins as antagonists.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics(8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 μM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous administration.

The proteins, antagonists, and agonists could be administered directlyto the host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8thed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosgae Forms: TabletsDekker, NY; and Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Disperse Systems Dekker, NY. The therapy of this invention may becombined with or used in association with other chemotherapeutic orchemopreventive agents.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. An isolated polypeptide comprising an amino acid sequence derivedfrom SEQ ID NO: 2, 4, 6, 8 or
 10. 2. The polypeptide of claim 1comprising the amino acid sequence of the mature protein.
 3. An isolatednucleic acid comprising a nucleotide sequence encoding an amino acidsequence derived from SEQ ID NO: 2, 4, 6, 8 or
 10. 4. The nucleic acidof claim 3 wherein the nucleotide sequence encodes the mature protein.5. The nucleic acid of claim 4 comprising the nucleotide sequence shownin SEQ ID: NO 1, 3, 5, 7 or
 9. 6. A fusion protein comprising thepolypeptide of claim
 1. 7. A binding compound which specifically bindsto the polypeptide of claim
 1. 8. The binding compound of claim 7 whichis an antibody or antibody fragment.
 9. The binding compound of claim 8wherein the antibody is a monoclonal antibody.
 10. An expression vectorcomprising the nucleic acid of claim
 3. 11. A host cell comprising thevector of claim
 10. 12. A process for recombinatly producing apolypeptide comprising culturing the host cell of claim 11 underconditions in which the polypeptide is expressed.
 13. A method fordetecting a specific nucleic acid sequence in a sample, said methodcomprising the steps of: (i) contacting a sample suspected to contain aspecific nucleic acid sequence with a probe comprising a nucleic acidsequence comprising at least 8 consecutive nucleotides selected from SEQID NO: 1, 3, 5, 7, or 9 under conditions in which a hybrid can formbetween said probe and the specific nucleic acid in said sample; and(ii) detecting any hybrid formed in step (i), wherein detection of saidhybrid indicates the presence of the specific nucleic acid sequence insaid sample.
 14. The method of claim 13 further comprising amplifyingsaid specific sequence in said sample prior to said detecting step. 15.A method for detecting a specific antigenic component in a sample, saidmethod comprising the steps of: (i) contacting a sample suspected tocontain a specific antigenic component encoded by an amino acid sequencederived from SEQ ID NO: 2, 4, 6, 8, or 10 with an antibody specific forsaid component, under conditions in which a stable antigen-antibodycomplex can form between said antibody and said antigenic component insaid sample; and (ii) detecting any antigen-antibody complex formed instep (i), wherein detection of an antigen-antibody complex indicates thepresence of said antigenic component in said sample.
 16. A method ofscreening for candidate therapeutic agents comprising: selecting as atarget sequence a polypeptide having an amino acid sequence derived fromSEQ ID NO: 2, 4, 6, 8 or 10; contacting a test compound with said targetsequence; and selecting as candidate therapeutic agent those testcompounds which bind to the target sequence.